Various severe ocular diseases are associated with an elevated intravitreal expression of VEGF-A which increases the permeability of retinal endothelial cells (REC) or retinal pigment epithelial (RPE) cells in vivo and in vitro. Inhibition of VEGF receptor 2 (VEGFR2) is sufficient to completely prevent VEGF-A165-induced dysfunctions of barriers formed by long-term cultivated, immortal human ARPE-19 cells or immortalized bovine retinal endothelial cells (iBREC). Extended exposure to VEGF-A could result in additional activation of other growth factor receptors, potentially promoting synergistic effects of corresponding factors on various cellular processes including angiogenesis. Based on these observations, we investigated whether blocking of VEGFR2 is also sufficient to revert VEGF-A-induced changes of the barriers consisting of iBREC (i.e. inner blood-retina barrier) or ARPE-19 cells (i.e. outer blood-retina barrier) in vitro. Alterations of confluent monolayers' properties induced by treatment with VEGF-A165 for one day followed by addition of small molecule inhibitors of the VEGFR2 were determined by continuous cell index (CI) measurements using the microelectronic biosensor system for cell-based assays xCELLigence. VEGF-A165 induced a long-lasting drop of the otherwise high CI of iBREC accompanied by reduced expression of the tight junction (TJ) protein claudin-1 and subtle changes of the plasma membrane localizations of TJ-protein claudin-5 and of vascular endothelial cadherin. Blocking mainly VEGFR2 with 10 nM nintedanib, 10 nM tivozanib or 500 nM ZM323881 efficiently reverted these changes within one day; higher concentrations of nintedanib or additional inhibition of neuropilin-1 were not superior. Interestingly, the CI of short-term cultivated, confluent ARPE-19 cells slightly increased in the presence of VEGF-A165, but was not changed by nintedanib. In contrast, VEGF-A165 markedly reduced the transepithelial electrical resistance of ARPE-19 cells cultivated on porous membrane inserts for three weeks, which was also accompanied by a significant loss of the then strongly plasma membrane-expressed TJ-protein ZO-1. These alterations were completely reverted within one day by 10 nM nintedanib of which higher concentrations were not superior. None of the inhibitors tested diminished the strong barrier properties of iBREC or long-term cultivated ARPE-19 cells. Taken together, inhibition of VEGFR2 efficiently reverts VEGF-A165-induced barrier disturbances of both cell types forming and regulating the inner and outer blood-retina barrier. As synergistic actions of growth factors seem to play only a minor role in inducing a barrier dysfunction, specific inhibition of VEGFR2 could be an interesting option to treat VEGF-A-induced macular edema without obvious effects on vitality and functions of REC and RPE cells.

中文翻译：

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VEGF受体2抑制剂nintedanib可以完全逆转VEGF-A165诱导的由视网膜内皮细胞或长期培养的ARPE-19细胞形成的屏障的干扰。

多种严重的眼部疾病与VEGF-A的玻璃体内表达升高有关，其在体内和体外增加了视网膜内皮细胞（REC）或视网膜色素上皮（RPE）细胞的通透性。对VEGF受体2（VEGFR2）的抑制足以完全预防VEGF-A165诱导的由长期培养的永生人类ARPE-19细胞或永生化牛视网膜内皮细胞（iBREC）形成的屏障功能障碍。长时间暴露于VEGF-A可能导致其他生长因子受体的额外激活，从而潜在地促进相应因子对包括血管生成在内的各种细胞过程的协同作用。基于这些观察，我们研究了对VEGFR2的阻断是否也足以逆转VEGF-A诱导的由iBREC组成的屏障的改变（即 内血视网膜屏障）或ARPE-19细胞（即外血视网膜屏障）。使用微电子生物传感器系统进行基于细胞的分析xCELLigence，通过连续细胞指数（CI）测量来确定通过用VEGF-A165处理一天，然后添加VEGFR2小分子抑制剂诱导的汇合单层性质的变化。VEGF-A165导致iBREC的高CI持续下降，伴随着紧密连接（TJ）蛋白claudin-1的表达降低以及TJ蛋白claudin-5和血管内皮细胞质膜定位的细微变化钙粘蛋白。用10 nM nintedanib，10 nM tivozanib或500 nM ZM323881主要阻断VEGFR2可以在一天之内有效地恢复这些变化。更高浓度的nintedanib或对神经纤溶蛋白1的其他抑制作用并不优越。有趣的是，在VEGF-A165存在的情况下，短期培养的，融合的ARPE-19细胞的CI略有增加，但nintedanib并没有改变。相比之下，VEGF-A165显着降低了在多孔膜插入物上培养的ARPE-19细胞经三周的跨上皮电阻，同时还伴随着明显丧失的强烈血浆膜表达的TJ蛋白ZO-1。这些变化在一天之内被10 nM nintedanib完全还原，后者的浓度更高。测试的抑制剂均未减弱iBREC或长期培养的ARPE-19细胞的强屏障特性。在一起 对VEGFR2的抑制可有效地逆转VEGF-A165诱导的形成和调节内，外血视网膜屏障的两种细胞类型的屏障障碍。由于生长因子的协同作用似乎仅在诱导屏障功能障碍中起次要作用，因此特异性抑制VEGFR2可能是治疗VEGF-A引起的黄斑水肿的有趣选择，而对REC和RPE细胞的活力和功能没有明显影响。